The Cephalic Sensory Organs of Acteon Tornatilis (Linnaeus, 1758) (Gastropoda Opisthobranchia) – Cellular Innervation Patterns As a Tool for Homologisation*

Home , Acteonidae, Acteonoidea, Acteon (gastropod), Acteon tornatilis, Cephalaspidea, Micromelo

Bonner zoologische Beiträge Band 55 (2006) Heft 3/4 Seiten 311–318 Bonn, November 2007

The cephalic sensory organs of Acteon tornatilis (Linnaeus, 1758) (Gastropoda Opisthobranchia) – cellular innervation patterns as a tool for homologisation*

Sid STAUBACH & Annette KLUSSMANN-KOLB1) 1)Institute for Ecology, Evolution and Diversity – Phylogeny and Systematics, J. W. Goethe-University, Frankfurt am Main, Germany

*Paper presented to the 2nd International Workshop on Opisthobranchia, ZFMK, Bonn, Germany, September 20th to 22nd, 2006

Abstract. Gastropoda are guided by several sensory organs in the head region, referred to as cephalic sensory organs (CSOs). This study investigates the CSO structure in the opisthobranch, Acteon tornatilis whereby the innervation patterns of these organs are described using macroscopic preparations and axonal tracing techniques. A bipartite cephalic shield and a lateral groove along the ventral side of the cephalic shield was found in A. tornatilis. Four cerebral nerves can be described innervating different CSOs: N1: lip, N2: anterior cephalic shield and lateral groove, N3 and Nclc: posterior cephalic shield. Cellular innervation patterns of the cerebral nerves show characteristic and constant cell clusters in the CNS for each nerve. We compare these innervation patterns of A. tornatilis with those described earlier for Haminoea hydatis (STAUBACH et al. in press). Previously established homologisation criteria are used in order to homologise cerebral nerves as well as the organs innervated by these nerves. Evolutionary implications of this homologisation are discussed. Keywords. Haminoea hydatis, Cephalaspidea, axonal tracing, homology, innervation patterns, lip organ, Hancock´s organ.

1. INTRODUCTION

Gastropoda are guided by several organs in the head re- phylogenetic position of Acteonoidea within Opistho- gion which are assumed to have primarily chemo- and branchia unsettled. Acteonoidea are characterised by the mechanosensory functions (AUDESIRK 1979; DAVIS & presence of a prominent cephalic shield. This structure is MATERA 1982; BICKER et al.1982; EMERY 1992; CHASE also present in Cephalaspidea and has been considered to 2000; CROLL et al. 2003). In Opisthobranchia, these be an apomorphie of the Cephalaspidea (SC H M E K E L 1 9 8 5 ) . cephalic sensory organs (CSOs) present an assortment of H o w e v e r, the structure of the cephalic shields differs con- forms including rhinophores, labial tentacles, oral veils, siderably in Cephalaspidea and Acteonoidea with the lat- Hancock´s organs and cephalic shields. Recent investiga- ter possessing two distinct hemispheres while the cephal- tions of CSOs in Opisthobranchia have focussed prima- ic shield in the Cephalaspidea possesses uniform structure. rily on functional aspects (CROLL 1983; BOUDKO et al. Therefore, common origin of both types of cephalic 1999; CROLL et al. 2003) while homology of the differ- shields and thus homology is questionable. Further CSOs ent types of CSOs in different taxa has never been inves- have been described in Acteonoidea and Cephalaspidea tigated in detail. We want to clarify their homology in sep- such as lip organ and Hancock´s organ (RUDMAN 1971A; arate evolutionary lineages so as to elucidate key ques- RUDMAN 1971B; RUDMAN 1972a, b; RUDMAN 1972c; tions regarding character evolution and phylogeny. EDLINGER 1980).

Acteon tornatilis belongs to the subgroup Acteonoidea, Since the presence of these organs in members of the formerly ascribed to the basal Cephalaspidea (ODHNER genus Acteon has been disputed by different authors 1939, BURN & THOMPSON 1998). However, recent inves- (EDLINGER 1980; SCHMEKEL 1985), absolute clarification tigations have either excluded the Acteonoidea from the is certainly necessary . The intention of this study is to de- Opisthobranchia (MIKKELSEN 1996) or proposed a sister scribe the structure emphasizing the innervation of the group relationship of Acteonoidea and the highly derived CSOs in the acteonid A. tornatilis. Our descriptions fo- Nudipleura (VONNEMANN et al. 2005) thus, rendering the cus on the cellular innervation patterns reconstructed for 312 Sid STAUBACH & Annette KLUSSMANN-KOLB: Cephalic sensory organ in Aceton the cerebral nerves using axonal tracing. In an earlier study ed on an objective slide dorsal side up in Entellan (VWR (STAUBACH et al. in press) these cellular innervation pat- International) and covered with a cover slip. Ten replicates terns were shown to be more adequate in homologising were prepared for each cerebral nerve of A. tornatilis. cerebral nerves than ganglionic origins of nerves (HUBER Samples with only a partial staining of the nerve were not 1993). By comparising the innervation patterns in A. tor - used because of possible incomplete innervation patterns. n a t i l i s to previously published data on H. hydatis Our criterion for a well-stained preparation was a dark blue (STA U B A C H et al. in pre s s), we want to survey whether the stained nerve indicating intact axons ( FR E D M A N 1 9 8 7 ). T h e preliminary characteristic cell clusters in the central nerv- Ni-Lys tracings were analysed by light microscopy (Le- ous system (CNS) of both taxa can be identified by ho- ica TCS 4D). Camera lucida drawings were digitalised fol- mologising cerebral nerves across taxa. Based on the ho- lowing the method of CO L E M A N ( 2 0 0 3 ) adapted for Corel- mologisation of the nerves innervating the CSOs we want DRAW 11. The somata in the innervation scemes occurs to clarify if A. tornatilis has homologous structures to the in all replicates. Somata only occurring in single samples CSOs of Cephalaspideans. It is our intent interest that we are not considered part of the schematics. The axonal path- shed light on the phylogenetic position and evolutionary ways are estimated over all replicates. Additionally, we history of Acteonoidea within the Opisthobranchia for fu- tested for asymmetries making axonal tracings (n = 2 to ture studies. 3) for each cerebral nerve of the left cerebral ganglion.

2.3. Scanning electron microscopy studies 2. MATERIALS AND METHODS The specimens were relaxed by an injection of 7 % Mg- 2.1. Specimens C l 2 in the foot. T h e r e a f t e r, the entire head region was dissected from the rest of the animal. The CSOs were fixed A. tornatilis (Fig. 1A) were collected in the wild at St. in 2,5 % glutaraldehyde, 1 % paraformaldehyde in 0,1M Michel en Greve (Brittany, France). They were then stored phosphate buffer (pH 7,2) at room temperature. For the alive at our lab in Frankfurt. Fourty specimens measur- SEM, the fixed CSOs were dehydrated through a graded ing a shell length between 15 and 20 mm were traced di- acetone series followed by critical point drying (CPD 030, rectly (5 to 15 days after collecting) and five were fixed BAL-TEC). Finally, they were spattered with gold (Sput- for SEM. ter-Coater, Agar Scientific) and examined with a Hitachi S4500 SEM. All photographs were taken using DISS (Dig- 2.2. Tracing studies ital Image Scanning System – Point Electronic) and subsequently adjusted for brightness and contrast with Corel Animals were relaxed with an injection of 7 % magne- PHOTO-PAINT 11. sium chloride. The central nervous system consisting of the cerebral, pleural and pedal ganglia was removed and placed in a small Petri dish containing filtered artificial 3. RESULTS seawater (ASW; Tropic Marin, Rebie-Bielefeld; GER- MANY). We then followed the procedures from CROLL 3.1. Organisation and innervation of the cephalic sen- & BAKER (1990) for Ni2+-lysine (Ni-Lys) tracing of ax- sory organs ons. Briefly, the nerves of the right cerebral ganglion were dissected free from the connective tissue. The nerves were A. tornatilis possesses a prominent bipartite cephalic shield cut and the distal tip was gently drawn into a glass mi- (cs) in which each hemisphere of this shield is divided in- cropipette using suction provided by an attached 2.5 ml to an anterior and a posterior lobe (Figs. 1A and B). Eyes syringe. Subsequently, the saline in the micropipette was are embedded deeply within the tissue of the shield. A l o n g replaced by a Ni-Lys solution (1.9g NiCl-6H2O, 3,5 g L- the lateral margin of the anterior lobe of the cephalic shield Lysine freebase in 20 ml double distilled H2O). The prepa- a groove is present (Fig. 1B, 2A). Hidden under the cs and ration was then incubated for 12–24 hours at 8º C to al- above the foot, the mouth opening is situated at the me- low transport of the tracer. The micropipette was then re- dian frontal edge (Fig. 2B) surrounded by the lip (not vis- moved and the ganglia were washed in ASW three times. ible in Figure 2B). We found four nerves innervating the The Ni-Lys was precipitated by the addition of five to ten CSOs (Fig.1B). The N1 (Nervus oralis) provides inner- drops of a saturated rubeanic acid solution in absolute Di- vation to the lip and small median parts of the anterior methylsulfoxide (DMSO). After 45 minutes the ganglia cephalic shield. The bifurcated N2 (Nervus labialis/labio- were transferred to 4 % paraformaldehyde (PFA) and fixed tentacularis) innervates the complete anterior cephalic for 4–12 hours at 4º C. Thereafter the ganglia were dehy- shield whereby the groove at the ventral anterior lobe of drated in an increasing ethanol series (70/80/90/99/99% the cephalic shield is especially innervated. The small N3 10 minutes each), cleared in methylsalicylate and mount- (Nervus tentacularis/rhinophoralis) innervates a little re- Bonner zoologische Beiträge 55 (2006) 313 gion of the posterior cephalic shield. The Nclc (Nervus For the N1 (n = 10) we identified six cerebral clusters clypei capitis) innervates the largest hind part of the pos- (Cnoc1-6) and one pedal cluster (Pdnoc1) in each sam- terior cephalic shield. We could not detect a lip organ (Fig. ple (Fig. 3A). The variation between the samples was re- 2B), which according to ED L I N G E R (1980) should comprise stricted to very few somata in some clusters. The cerebral two small lobes on the cephalic shield above the mouth. clusters were distributed over the whole cerebral ganglion. A Hancock´s organ described by EDLINGER (1980) for A. The pedal cluster Pdnoc1 was located on the anterior mar- Tornatilis, here a folded structure separated from the gin of the pedal ganglion above the pedal commissure. T h e cephalic shield was likewise not found in the present study. innervation pattern of the N2 (n=10) consists of five cerebral clusters (Cnlc1-5) and three pedal clusters (Pdnlc1- 3.2. Tracing studies 3) (Fig. 3B). The cerebral clusters show distinct spatial separations and are easy to identify. The third traced cere- By conducting the axonal tracing studies we were able to bral nerve (n = 10) was the N3. Six cerebral (Cnrc1-6) and reconstruct cellular innervation patterns for the four cere- three pedal clusters (Pdnrc1-3) were identified (Fig. 3C). bral nerves of A. tornatilis. Ten replicate tracings were per- We found an additional single cluster (C c l n rc 1) and a sin- formed each for the N1 N2, N3 and Nclc using only the gle soma in the left cerebral ganglion (see arrows in Fig. nerves of the right cerebral ganglion. The characteristic 3C). The contralateral cluster was located at the base of patterns of labelled somata for all nerves are shown in Fig- the N2 whereas the single soma was found at the root of ure 3A-D, including the approximate pathways of the the cerebral commissure. We observed slight intraspecif- stained axons. The identified clusters were named with ab- ic variability between the ten samples which amounted on- breviations signifying the ganglion in which they are lo- ly to very few somata in some clusters. In the Nclc, the cated, the nerve filled and a number indicating the order innervation (n = 10) pattern consisted of five cerebral clus- of their description (for example, Cnlc3: Cerebral Nervus ters (Cncc1-5) and a single soma at the lateral margin of labialis cluster 3). Nerve cells are grouped in clusters on the cerebral ganglion above the pedal connective (Fig. the basis of their close positioning in the ganglia and the 3D). Additionally we found four pedal clusters (Pdncc1- tight fasciculation of their axons projecting into the filled 4). The Nclc had the highest amount of pedal clusters in nerve. Asymmetries for tracings of the left nerves could all investigated nerves. The number of pedal somata, how- not be detected. ever, was comparable to the number of pedal somata for the N2 innervation pattern (Fig 3B).

Fig. 1. A: Photograph of Acteon tornatilis with the cephalic shield visible. B: Schematic illustration of the CNS, the four cerebral nerves (excluding the optical nerve) and the cephalic sensory organs of Haminoea hydatis and Acteon tornatilis. Only the right cerebral nerves are shown. N1 Nervus oralis, N2 Nervus labialis, N3 Nervus rhinophoralis, Nclc Nervus clypei capitis, ey eye, gr groove, al anterior lobe, pl posterior lobe, sh shell, cs cephalic shield, f foot. 314 Sid STAUBACH & Annette KLUSSMANN-KOLB: Cephalic sensory organ in Aceton

Fig. 2. A. Lateral SEM photography of the groove at the ventral surface of the cephalic shield of Acteon tornatilis. cs cephalic shield, gr groove. B. Frontal SEM photography of the mouth region of Acteon tornatilis. cs – cephalic shield, mo – mouth, f – foot.

4. DISCUSSION missing in A. tornatilis. Hence, we expected considerable differences in the cellular innervation patterns for the N3 The present study demonstrates the constancy of nervous of these species. However, these differences were marg i n- structures in the opisthobranch mollusc. Throughout our al and only amounted to the lack of one single cell soma investigation of several individuals of the acteonid, A c t e o n in the cerebral ganglion of A. tornatilis. This implies that tornatilis we found uniform innervation patterns of the basic innervation patterns of the N3 are probably the same head region via four cerebral nerves, which can be attrib- in both species. Additional functions of the N3 processed uted to characteristic neuronal cell clusters in the CNS. in the rhinophoral ganglion can be proposed for H. hydatis. These cellular innervation patterns in A. tornatilis show These functions are probably related to the Hancock´s or- an extremely high congruence with the cellular innerva- gan, which is innervated by nerves originating in the tion patterns described for the four cerebral nerves of rhinophoral ganglion (STA U B A C H et al. in pre s s). We were Haminoea hydatis (STAUBACH et al. in press). unable to locate such an organ in A. tornatilis in contrast to earlier descriptions (EDLINGER 1980). In the N1, the number of cerebral clusters as well as the position of these clusters to each other is the same in A. Upon comparing the innervation patterns presented here tornatilis and H. hydatis. However, we found some dif- for A. tornatilis with those for H. hydatis (STAUBACH et ferences in the size and number of somata when compar- al. in press) we find constant features of these patterns ing both species. A d d i t i o n a l l y, we could not detect a pleu- across species. This is congruent with other findings that ral, a parietal and a pedal cluster in A. tornatilis, which neuronal structures in the central nervous system of mol- were described for H. hydatis. This may be due to the dif- luscs and other invertebrates seem to be highly conserved ferences in the peripheral innervation area of the N1. In (CROLL 1987; ARBAS 1991; HAYMAN-PAUL 1991; KUTSCH A. tornatilis it only provides for the lip and very small parts & BREIDBACH 1994; NEWCOMB et al. 2006). Hence, we of the median cephalic shield whereas in H. hydatis, it in- postulate the N1 of A. tornatilis to be homologous to the nervates the lip and large parts of the anterior cephalic N1 (Nervus oralis) described by HU B E R ( 1 9 9 3 ) for Cepha- shield. For the second nerve, the N2 (Nervus labialis), we laspideans. Additionally, we postulate homologies of the nearly found no differences between the presence and dis- N2 and the N3 of A. tornatilis to the N2 (Nervus labialis) tributions of the cell clusters for both species. The only and N3 (Nervus rhinophoralis) of Chepalaspideans. This ostentatious difference was the lack of a single pedal so- is congruent to the assumption of HOFFMANN (1939) that ma and its contra-lateral analogue in A. tornatilis. In the the c3 (after VAYSSIÈRE 1880) of H. hydatis represents the Nclc (Nervus clypei capitis), the difference between the Nervus labialis and the c4 represents the Nervus tentac- two species was also reduced to the presence of a single ularis, here a synonym for the Nervus rhinophoralis (HU- cerebral soma in A. tornatilis. In contrast to the three B E R 1 9 9 3 ). Our data cannot support ED L I N G E R’S ( 1 9 8 0 ) d e- nerves described above, we found a prominent difference scription of independent nerves for the lip organ (N1 af- in the structure of the N3 when comparing Acteon and ter Edlinger 1980) and the anterior Hancock`s organ (N2 Haminoea. On the other hand, in H. hydatis the N3 ter- after Edlinger 1980). The Nclc of A. tornatilis also seems minates in a rhinophoral ganglion. Such a ganglion is to be homologous to the Nclc of Cephalaspideans (Huber Bonner zoologische Beiträge 55 (2006) 315

Fig. 3. Schematic outline of cell clusters providing the N1 (A), N2 (B), N3 (C) and Nclc (D) of Acteon tornatilis. The size and position of the somata were digitalized from a camera lucida drawing, the distribution of the axons are averaged from all replicates. N1 Nervus oralis, N2 Nervus labialis, N3 Nervus rhinophoralis, Nclc Nervus clypei capitis, N. opt. Nervus opticus, CG cerebral ganglia, RhG rhinophoral ganglia, PlG pleural ganglia, PdG pedal ganglia. 316 Sid STAUBACH & Annette KLUSSMANN-KOLB: Cephalic sensory organ in Aceton

1993). HO F F M A N N (1939) described the same nerve as the datis; and 3. the posterior Hancock´s organ has second- Nervus proboscidis. We define this nerve however, as arily been reduced in A. tornatilis. Nervus clypei capitis according to HUBER (1993). The first hypothesis is rather implausible since we found Considering the homologisation of the cerebral nerves in a distinct N3 with conserved cellular innervation patterns light of their neurological origin, neuro-anatomics and in the central nervous system. If the ancestor of A. tor- nervous innervation patterns, we postulate hypotheses of natilis never had a posterior Hancock´s organ, this nerve homologies respective of the organs innervated by these and associated neural structures should be lacking. More- nerves. Thus, we consider the lip of A. tornatilis to be ho- over, a Hancock´s organ has been described for other mologous to the lip of Cephalaspideans (HUBER 1993) Acteonoidea (RU D M A N 1 9 7 1a, b; RU D M A N 1972a, b; RU D- since both organs are innervated by the N1. The same MAN 1972c). If we consider the second explanation for holds true for the small median parts of the cephalic shield lack of a posterior Hancock´s organ in A. tornatilis, we in Acteon and the anterior cephalic shield of Haminoea. imply that the posterior cephalic shield in this species, in- We could not find a lip organ in A. tornatilis as described nervated by the N3, presents a sensory organ as the Han- by EDLINGER (1980), but we detected a groove at the ven- cock´s organ in Cephalaspidea. However, immunohisto- tral side of the anterior cephalic shield. This groove is in- chemical and ultrastructural investigations of the respec- nervated by the N2 as is the lip organ of Cephalaspideans tive epithelia in A. tornatilis do not indicate a sensory func- (HUBER 1993). Therefore, we postulate this groove to be tion at all (S. FALLER, Frankfurt, pers. comm. 2007; homologous to the lip organ. This hypothesis is also sup- GÖBBELER & KLUSSMANN-KOLB in press). We reject this ported by data on immunoreactivity against several neu- hypothesis of homology of the posterior cephalic shield rotransmitters. In the groove of A. tornatilis as well as in in A. tornatilis and posterior Hancock´s organ in H. hy - the lip organ of H. hydatis, characteristic sub-epidermal d a t i s since we found no evidence for a function of the pos- sensory neurons containing catecholamines could be found terior cephalic shield as an olfactory sensory organ. More- in high density indicating that both organs are involved o v e r, the posterior cephalic shield is mostly innervated by in contact chemoreception (S. FALLER, Frankfurt, pers. the Nclc and not by the N3. The third hypothesis regard- comm. 2007). ing the reduction of a Hancock´s organ seems to be the most plausible when the habitat and the food sources of The N2 of Haminoea is divided into two branches which A. tornatilis in comparison to H. hydatis are considered. are described as two single nerves by EDLINGER (1980). The posterior Hancock´s organ is believed to be an olfac- The first or inner branch provides the lip organ as de- tory sensory organ (AU D E S I R K 1979; EM E RY 1 9 9 2). H. hy - scribed earlier. The second, outer branch is related to the datis feeds on green algae which occur in patches in open anterior Hancock´s organ ( E D L I N G E R 1980; HU B E R 1 9 9 3 ). water whereas A. tornatilis is a predator of soft inverte- In Acteon we also found two branches of the N2: the in- brates living up to ten centimeters in solid sand (FRETTER ner one providing the largest part of the groove whereas 1939; YONOW 1989; own investigations). In such an en- the outer branch is restricted to a small region between vironment, an olfactory sensory organ is not plausible the anterior and posterior lobe of the cephalic shield. since olfaction or distance chemoreception is generally as- Therefore, this latter region may be homologous to the an- sociated with water currents, which are not substantial in terior Hancock´s organ of H. hydatis and not to the pos- a sandy substrate habitat. Here, a contact chemoreceptor, terior Hancock´s organ as described by EDLINGER (1980). which is located near the edge of the cephalic shield is more plausible. This we witnessed in Acteon tornatilis v i a The N3 of A. tornatilis provides a large part of the pos- its display of a potentially chemoreceptive groove along terior cephalic shield but no identifiable posterior Han- the lateral margin of the anterior cephalic shield. cock´s organ. Additional immunohistochemical and ultrastructural investigations could also not detect a posterior This assumption of secondary reduction of the Hancock´s Hancock´s organ in A. tornatilis (S. FALLER, Frankfurt, o rgan in the endobenthic A. tornatilis is also supported by pers. comm. 2007; GÖBBELER & KLUSSMANN-KOLB in the fact that a Hancock´s organ has been described for oth- p re s s). The posterior parts of the cephalic shields in A c t e o n er epibenthic Acteonoidea (e. g. Bullina, Micromelo, Hy - and Haminoea are probably equally homologous as both datina) (RUDMAN 1971a, b; RUDMAN 1972a, b; RUDMAN where innervated by the Nclc. 1972a,b,c).

The lack of a posterior Hancock´s organ in A. tornatilis Despite all discussion, homology of the described Han- might be due to three different reasons: 1. the ancestor of cock´s organs to those in Cephalaspidea cannot undoubt- A. tornatilis never had a posterior Hancock´s organ; 2. the edly be proposed at this stage, particularly since data on posterior cephalic shield of A. tornatilis may be a homol- innervation patterns in these acteonids are lacking to date. ogous structure to the posterior Hancock´s organ of H. hy - M o r e o v e r, current phylogenetic hypotheses (GR A N D E et al. Bonner zoologische Beiträge 55 (2006) 317

CR O L L, R. P., BO U D K O, D. Y., PI R E S , A. & HA D F I E L D, M. G. 2003. 2004; VO N N E M A N N et al. 2005) regarding Opistho- Transmitter content of cells and fibers in the cephalic senso- branchia propose an independent origin of Acteonoidea ry organs of the gastropod mollusc Phestilla sibogae. Cell Ti s- and Cephalaspidea, indicating convergent development of sue Research 314: 437–448. these sensory organs in both evolutionary lineages. Fur- DAV I S, W. J. & MAT E R A, E. M. 1982. Chemoreception in gastro- ther studies will have us utilizing cellular innervation pat- pod molluscs: electron microscopy of putative receptor cells. terns for CSOs in order to compare several taxa while ho- Journal of Neurobiology 13(1): 79–84. EDLINGER, K. 1980. Zur Phylogenie der chemischen Sinnesor- mologising the different types of CSOs in Opistho- gane einiger Cephalaspidea branchia. This procedure will enable us to glean a better (Mollusca, Opisthobranchia). 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